Improved Simulated-Daylight Photodynamic Therapy and Possible Mechanism of Ag-Modified TiO2 on Melanoma

Simulated-daylight photodynamic therapy (SD-PDT) may be an efficacious strategy for treating melanoma because it can overcome the severe stinging pain, erythema, and edema experienced during conventional PDT. However, the poor daylight response of existing common photosensitizers leads to unsatisfactory anti-tumor therapeutic effects and limits the development of daylight PDT. Hence, in this study, we utilized Ag nanoparticles to adjust the daylight response of TiO2, acquire efficient photochemical activity, and then enhance the anti-tumor therapeutic effect of SD-PDT on melanoma. The synthesized Ag-doped TiO2 showed an optimal enhanced effect compared to Ag-core TiO2. Doping Ag into TiO2 produced a new shallow acceptor impurity level in the energy band structure, which expanded optical absorption in the range of 400–800 nm, and finally improved the photodamage effect of TiO2 under SD irradiation. Plasmonic near-field distributions were enhanced due to the high refractive index of TiO2 at the Ag-TiO2 interface, and then the amount of light captured by TiO2 was increased to induce the enhanced SD-PDT effect of Ag-core TiO2. Hence, Ag could effectively improve the photochemical activity and SD-PDT effect of TiO2 through the change in the energy band structure. Generally, Ag-doped TiO2 is a promising photosensitizer agent for treating melanoma via SD-PDT.


Introduction
Melanoma is a commonly occurring severe skin malignancy induced by melanocytes [1]. The incidence of melanoma is ever-increasing. It is traditionally considered to be metastatically invasive as it can invade and spread to neighboring tissues [2]. Additionally, it is resistant to chemotherapeutic drugs and radiation therapy [3,4]. Therefore, more effective therapeutic strategies for melanoma need to be developed [5,6]. Photodynamic therapy (PDT) can selectively destroy diseased cells or tissues as they are more sensitive to light irradiation [7]. During PDT, a photosensitizer in a singlet ground state undergoes visible or near-infrared irradiation, absorbs energy, and attains an excited triplet state through intersystem crossing [8]. The triplet state of the photosensitizer reacts with oxygen or the substrate through electron/hydrogen atom or energy transfer processes, producing reactive oxygen species, especially singlet oxygen, to damage biological components (e.g., amino acids, unsaturated lipids, and DNA bases) [9]. Because singlet oxygen can diffuse by only 10-20 nm during its lifetime of 0.01-0.04 µs, the intracellular damage targets of PDT are very close to the intracellular localization of the photosensitizer. Therefore, PDT might be a non-invasive, effective treatment strategy for melanoma cancer therapy [10].
PDT efficiency relies on three primary factors: the photosensitizer, light, and molecular oxygen, as per the PDT mechanism [11,12]. The frequently utilized light sources for PDT include coherent light sources (argon and argon-pumped lasers, solid-state lasers, metal vapor-pumped dye lasers, and optical parametric oscillator lasers) as well as non-coherent

Synthesis and Characterization of Ag-Modified TiO 2 with Different Structure
To increase the light response range of TiO 2 and improve the simulated-daylight PDT effect of TiO 2 on melanoma, Ag-modified TiO 2 was synthesized. Based on the different optical properties of nanomaterials with different structures, Ag-doped TiO 2 and Ag-core TiO 2 were synthesized, respectively. The TEM results of the synthesized TiO 2 were comparable to P25 (the commercialized TiO 2 nanoparticles) purchased from Sigma, and Ag-doped TiO 2 showed that the size of the sphere was 100 nm ( Figure 1A,B). Nonetheless, the absorption spectrum of the Ag-doped TiO 2 displayed a prominent redshift (Figure 2A), which may be attributed to alterations in the energy band structure. The XRD findings demonstrated that the lattice structure of TiO 2 remained unaltered following Ag being doped into TiO 2 ( Figure 2B). In Ag-core TiO 2 , the addition of sodium bicarbonate to the reaction mixture led to the formation of a TiO 2 shell enveloping Ag nanoparticles. The crystal lattice structure of TiO 2 with a spacing of 0.32 nm appeared after calcination ( Figure 1C,D). The TEM results revealed that the synthesized silver had a uniform particle size ( Figure 1E). The thickness of the shell of TiO 2 increased with the increase in the concentration of sodium bicarbonate to 1.5 mL. When the concentration of sodium bicarbonate was 0.9 mL and 1.3 mL, the thickness of the TiO 2 shell was about 5 nm and 18.7 nm, respectively ( Figure 1F,G).
When the concentration of sodium bicarbonate was 1.5 mL, the shell of TiO 2 agglomerated ( Figure 1H). The results of the energy spectrum analysis from TEM-EDS also confirmed that silver was successfully coated by TiO 2 (Ag 81.43%, Ti 7.36%, and O 11.21%) ( Figure 2D). The results of the absorption spectrum showed that the Ag-doped TiO 2 showed absorption in the range of 400-800 nm, which was similar to the absorption of the synthesized TiO 2 and higher than the absorption of P25 (Figure 2A). The Ag-core TiO 2 showed a prominent red shift and a decrease in the intensity of the absorption spectrum with an increase in the thickness of the TiO 2 shell ( Figure 2C). ure 1C,D). The TEM results revealed that the synthesized silver had a uniform particle size ( Figure 1E). The thickness of the shell of TiO2 increased with the increase in the concentration of sodium bicarbonate to 1.5 mL. When the concentration of sodium bicarbonate was 0.9 mL and 1.3 mL, the thickness of the TiO2 shell was about 5 nm and 18.7 nm, respectively ( Figure 1F,G). When the concentration of sodium bicarbonate was 1.5 mL, the shell of TiO2 agglomerated ( Figure 1H). The results of the energy spectrum analysis from TEM-EDS also confirmed that silver was successfully coated by TiO2 (Ag 81.43%, Ti 7.36%, and O 11.21%) ( Figure 2D). The results of the absorption spectrum showed that the Ag-doped TiO2 showed absorption in the range of 400-800 nm, which was similar to the absorption of the synthesized TiO2 and higher than the absorption of P25 (Figure 2A). The Ag-core TiO2 showed a prominent red shift and a decrease in the intensity of the absorption spectrum with an increase in the thickness of the TiO2 shell (Figure2C). Transmission electron microscopy image of Ag-core TiO2 with a high resolution before the calcination treatment; (D) The crystal lattice structure of TiO2 in Ag-core TiO2 observed using transmission electron microscopy image with a high resolution after the calcination treatment; (E) Transmission electron microscopy image of Ag; (F) Transmission electron microscopy image of Ag-core TiO2 with 5 nm thick TiO2 shell (sodium bicarbonate at 0.9 mL); (G) Transmission electron microscopy image of Ag-core TiO2 with 20 nm thick TiO2 shell (sodium bicarbonate at 1.3 mL); (H) Transmission electron microscopy image of Ag-core TiO2 with shell of TiO2 agglomerated (sodium bicarbonate at 1.5 mL). (C) Transmission electron microscopy image of Ag-core TiO 2 with a high resolution before the calcination treatment; (D) The crystal lattice structure of TiO 2 in Ag-core TiO 2 observed using transmission electron microscopy image with a high resolution after the calcination treatment; (E) Transmission electron microscopy image of Ag; (F) Transmission electron microscopy image of Ag-core TiO 2 with 5 nm thick TiO 2 shell (sodium bicarbonate at 0.9 mL); (G) Transmission electron microscopy image of Ag-core TiO 2 with 20 nm thick TiO 2 shell (sodium bicarbonate at 1.3 mL); (H) Transmission electron microscopy image of Ag-core TiO 2 with shell of TiO 2 agglomerated (sodium bicarbonate at 1.5 mL).

Comparative Analysis of the Photocatalytic Activity of Ag-Modified TiO 2 with Different Structures
The photochemical activities of Ag-modified TiO 2 were evaluated through the photocatalytic degradation of methylene blue. As shown in Figure 3A, methylene blue could be degraded under TiO 2 induction after Ag was added to Ag-doped TiO 2 , but this degradation effect was not observed for free TiO 2 . Upon reaching a 2% Ag concentration, the degradation rate rose from 40% to 70%. As shown in Figure 3B, methylene blue was degraded by 62.3% and 36.3% under the induction of Ag-core TiO 2 (5 nm thick) and P25, respectively. The catalytic efficiency of Ag-core TiO 2 decreased as the thickness of the shell increased, and the Ag-core TiO 2 (5 nm thick) of the shell had the highest catalytic efficiency. Hence, Ag-modified TiO 2 significantly increased the photochemical activity of TiO 2 . Compared to Ag-core TiO 2 , Ag-doped TiO 2 exhibited better photochemical activity.

Comparative Analysis of the Photocatalytic Activity of Ag-Modified TiO2 with Different Structures
The photochemical activities of Ag-modified TiO2 were evaluated through the photocatalytic degradation of methylene blue. As shown in Figure 3A, methylene blue could be degraded under TiO2 induction after Ag was added to Ag-doped TiO2, but this degradation effect was not observed for free TiO2. Upon reaching a 2% Ag concentration, the degradation rate rose from 40% to 70%. As shown in Figure 3B, methylene blue was degraded by 62.3% and 36.3% under the induction of Ag-core TiO2 (5 nm thick) and P25, respectively. The catalytic efficiency of Ag-core TiO2 decreased as the thickness of the shell increased, and the Ag-core TiO2 (5 nm thick) of the shell had the highest catalytic efficiency. Hence, Ag-modified TiO2 significantly increased the photochemical activity of TiO2. Compared to Ag-core TiO2, Ag-doped TiO2 exhibited better photochemical activity.

Comparative Analysis of the Photocatalytic Activity of Ag-Modified TiO2 with Different Structures
The photochemical activities of Ag-modified TiO2 were evaluated through the photocatalytic degradation of methylene blue. As shown in Figure 3A, methylene blue could be degraded under TiO2 induction after Ag was added to Ag-doped TiO2, but this degradation effect was not observed for free TiO2. Upon reaching a 2% Ag concentration, the degradation rate rose from 40% to 70%. As shown in Figure 3B, methylene blue was degraded by 62.3% and 36.3% under the induction of Ag-core TiO2 (5 nm thick) and P25, respectively. The catalytic efficiency of Ag-core TiO2 decreased as the thickness of the shell increased, and the Ag-core TiO2 (5 nm thick) of the shell had the highest catalytic efficiency. Hence, Ag-modified TiO2 significantly increased the photochemical activity of TiO2. Compared to Ag-core TiO2, Ag-doped TiO2 exhibited better photochemical activity.

Comparative Analysis of the Cytotoxicity Assay and Phototoxicity of Ag-Modified TiO 2 with Different Structures
To evaluate the safety and photodamage effects of the synthesized Ag-doped TiO 2 and Ag-core TiO 2 , we determined the viability of A375 cells (human melanoma cell line) without irradiation and with irradiation, by performing a CCK-8 assay. Cell viability induced by 50 µg/mL TiO 2 in different reagents was higher than 90%, and no noticeable difference was found between them, which indicated that the synthesized reagents had negligible dark cytotoxicity in A375 cells. When the concentration of TiO 2 decreased, the cell viability increased further ( Figure 4A). Hence, the synthesized Ag-doped TiO 2 and Ag-core TiO 2 were safe for usage. As shown in Figure 4B, after irradiation by daylight, 1 µg/mL TiO 2 in the different reagents could not inhibit A375 cells, and 50 µg/mL TiO 2 in the different reagents could strongly inhibit them. Specifically, the simulated-daylight photodamage effect of Ag-doped TiO 2 was higher than that of Ag-core TiO 2 . For example, cell viability decreased to 24.5% and 31.6% after induction by Ag-doped TiO 2 and Agcore TiO 2 , respectively. However, cell viability only decreased to 67.6% and 60.1% after induction by P25 and the synthesized free TiO 2 at 50 µg/mL. Ag-core TiO2 were safe for usage. As shown in Figure 4B, after irradiation by daylight, 1 μg/mL TiO2 in the different reagents could not inhibit A375 cells, and 50 μg/mL TiO2 in the different reagents could strongly inhibit them. Specifically, the simulated-daylight photodamage effect of Ag-doped TiO2 was higher than that of Ag-core TiO2. For example, cell viability decreased to 24.5% and 31.6% after induction by Ag-doped TiO2 and Ag-core TiO2, respectively. However, cell viability only decreased to 67.6% and 60.1% after induction by P25 and the synthesized free TiO2 at 50 μg/mL.
The photodamage effect increased with the increase in the irradiation dosage ( Figure  3C). However, after irradiation with 40 J/cm 2 , the untreated cells could also be partially inhibited, and the cell survival rate of A375 was only about 71%. Based on the principle of little toxic effect on normal tissue occurring to the greatest extent during PDT, the irradiation dosage for the synthesized Ag-modified TiO2 could be controlled below 40 J/cm 2 . Overall, Ag could effectively improve the photodamage effect of TiO2 under simulateddaylight irradiation, and Ag-doped TiO2 had a more significant daylight PDT effect on melanoma. Cell viability without irradiation-the cytotoxicity assay of Ag-modified TiO2 compared with P25 and the synthesized TiO2; (B) Cell viability after irradiation by daylight-the phototoxicity assay of Ag-modified TiO2 compared with P25 and the synthesized TiO2 with different concentrations of TiO2; (C) Cell viability after different irradiation dosage-the phototoxicity assay of Ag-modified TiO2 compared with P25 and the synthesized TiO2. *, p < 0.05, represents statistically significant difference between P25, the synthesized TiO2, Ag-core TiO2, the Ag-doped TiO2 group, and the control group.

Comparative Analysis of ROS Generation by Ag-Modified TiO2 with Different Structures
During PDT, generated ROS can lead to the damage of cellular components and then induce cell death. Hence, the ability to generate ROS determines the PDT effect. To determine the simulated-daylight PDT effect induced by Ag-modified TiO2, the ability to generate ROS was evaluated using a DCFH-DA probe-the most widely used probe for detecting intracellular H2O2 and oxidative stress. As per the fluorescence imaging findings, ROS were generated after induction by P25, the synthesized TiO2, and Ag-modified TiO2, and the induction was higher when Ag-modified TiO2 was used ( Figure 5A). The fluorescence intensity of DCFH-DA increased obviously in Ag-modified TiO2 ( Figure 5B). (C) Cell viability after different irradiation dosage-the phototoxicity assay of Ag-modified TiO 2 compared with P25 and the synthesized TiO 2 . *, p < 0.05, represents statistically significant difference between P25, the synthesized TiO 2 , Ag-core TiO 2 , the Ag-doped TiO 2 group, and the control group.
The photodamage effect increased with the increase in the irradiation dosage ( Figure 3C). However, after irradiation with 40 J/cm 2 , the untreated cells could also be partially inhibited, and the cell survival rate of A375 was only about 71%. Based on the principle of little toxic effect on normal tissue occurring to the greatest extent during PDT, the irradiation dosage for the synthesized Ag-modified TiO 2 could be controlled below 40 J/cm 2 . Overall, Ag could effectively improve the photodamage effect of TiO 2 under simulated-daylight irradiation, and Ag-doped TiO 2 had a more significant daylight PDT effect on melanoma.

Comparative Analysis of ROS Generation by Ag-Modified TiO 2 with Different Structures
During PDT, generated ROS can lead to the damage of cellular components and then induce cell death. Hence, the ability to generate ROS determines the PDT effect. To determine the simulated-daylight PDT effect induced by Ag-modified TiO 2 , the ability to generate ROS was evaluated using a DCFH-DA probe-the most widely used probe for detecting intracellular H 2 O 2 and oxidative stress. As per the fluorescence imaging findings, ROS were generated after induction by P25, the synthesized TiO 2 , and Ag-modified TiO 2 , and the induction was higher when Ag-modified TiO 2 was used ( Figure 5A). The fluorescence intensity of DCFH-DA increased obviously in Ag-modified TiO 2 ( Figure 5B). Compared with P25, Ag-core TiO 2 and Ag-doped TiO 2 resulted in significant increases, at average values of 1.75-fold and 1.95-fold, respectively. Hence, the ROS levels induced by Ag-doped TiO 2 were higher than those induced by Ag-core TiO 2 . The inhibitory activity induced by all reagents containing TiO 2 could be effectively weakened by using a quenching agent of ROS. After being treated with P25, the synthesized TiO 2 , Ag-core TiO 2 , and Ag-doped TiO 2 , as well as 20 mM histidine, cell activities increased from 67.6%, 60%, 31.6%, and 24.5% to 85.3%, 80.8%, 60.2%, and 55.6%, respectively. This revealed that the degree of inhibition induced by Ag-modified TiO 2 was higher than that induced by the synthesized TiO 2 or P25 ( Figure 5C). Hence, our findings showed that Ag-modified TiO 2 , especially Ag-doped TiO 2 , efficiently improved simulated-daylight PDT by enhancing the ability to generate ROS.
weakened and the ability of the TiO2 shell to capture light decreased with an increase in the thickness of the shell, which occurred probably because the shell affected the movement of photo-generated electrons and holes ( Figure 8C). Hence, the Ag-core TiO2 with a 5 nm thick shell had the highest photocatalytic efficiency. The bar is 20 μm or SD. *, p < 0.05, represents statistically significant difference between the treated histidine group and the untreated histidine group in P25, the synthesized TiO2, Ag-core TiO2, Agdoped TiO2, and control. The bar is 20 µm or SD. *, p < 0.05, represents statistically significant difference between the treated histidine group and the untreated histidine group in P25, the synthesized TiO 2 , Ag-core TiO 2 , Ag-doped TiO 2 , and control.

The Theoretical Mechanistic Analysis
The results of the experiment showed that modification with Ag could effectively enhance the photodamage effect of TiO 2 under simulated-daylight irradiation. Specifically, Ag-doped TiO 2 had a more significant daylight PDT effect on melanoma. This might be due to an increase in the response of TiO 2 to daylight facilitated by Ag. To confirm whether this mechanism was used, changes in the optical absorption properties of TiO 2 induced by doping with Ag were used, based on density functional theory using the CASTEP code. First, a supercell of TiO 2 (containing 16 Ti atoms and 32 O atoms) and 2% Ag-doped TiO 2 (containing 15 Ti atoms, 32 O atoms and 1 Ag atom) was constructed and used as the later calculation model ( Figure 6A,B). Then, the changes in the band structure and the density of the states of pure TiO 2 and Ag-doped TiO 2 were calculated. The energy of the band gap of pure TiO 2 was 3.325 eV, which then decreased to 3.14 eV when Ag was doped ( Figure 6C,D). Additionally, compared to the band structure of pure TiO 2 , two new impurity energy levels were introduced, and one of them passed through the Fermi level, which indicated that it can act as an acceptor at the shallow impurity energy level as a bound state of a hole ( Figure 6C,D). This shallow acceptor impurity level could enhance the separation of electron-hole pairs, produce free conduction holes, decrease the recombination of photo-generated electrons and holes, and decrease the energy required for the electrons to escape. The computed results of the total density of states and partial density of states of pure TiO 2 and Ag-doped TiO 2 showed that the 4d-orbital electrons of Ag atoms were used to introduce the new impurity energy levels and produce the new electronic states through strong mixing with the p-orbital states of oxygen atoms (Figure 7). Hence, these two impurity energy levels of Ag-doped TiO 2 mainly occurred due to doping with Ag. Generally, the doped Ag atom has an influence on the TiO 2 energy structure and induced bandgap narrowing. Ag is the main reason for the the increase in the daylight   First principles analysis can be used to analyze the properties of materials with a crystal structure but not of materials with a core-shell structure. To determine the cause of the improvement in the SD-PDT effect of TiO 2 induced by Ag-core TiO 2 , discrete dipole approximation simulations were studied because these changes might be induced by the localized surface plasmon resonance enhancement effect. First, a series of Ag-core TiO 2 complexes with a constant Ag core and TiO 2 shells of different thicknesses (0 nm, 5 nm, 10 nm, 15 nm, and 20 nm) were calculated. As shown in Figure 8A, the results showed that the absorption spectrum has an obvious red shift and a significant decrease in the intensity with an increase in the thickness of the TiO 2 shell. These results matched the absorption spectrum data we measured. Plasmonic near-field distribution showed a strong change in the Ag-core TiO 2 ( Figure 8B). The electric near-field intensities were considerably enhanced due to the high refractive index of TiO 2 at the Ag-TiO 2 interface. With the increase in the electric near-field intensity, the amount of light captured by TiO 2 and the PDT effect increased. The relationship between the field enhancement effect and the thickness of the TiO 2 shell results showed that the field enhancement effect gradually weakened and the ability of the TiO 2 shell to capture light decreased with an increase in the thickness of the shell, which occurred probably because the shell affected the movement of photo-generated electrons and holes ( Figure 8C). Hence, the Ag-core TiO 2 with a 5 nm thick shell had the highest photocatalytic efficiency.

Discussion
Photodynamic therapy is an effective therapeutic strategy for skin disease in clinical therapy [38]. However, it is often accompanied by severe adverse effects during treatment. A large number of patients are unable to continue treatment due to these adverse effects. In 2008, daylight PDT was first introduced as a less painful, outdoors alternative to conventional PDT, with similar clinical effectiveness [39]. However, daylight PDT efficacy was often dependent on weather conditions. For example, in the U.K., daylight PDT is practical between the months of March or April and September or October, when the temperature is above 10 °C in the day (from 9:00 to 18:00) and the fluence rate reaches 130 W/m 2 [40]. In addition, to avoid patient exposure to harmful wavelengths of ultraviolet radiation during daylight PDT, organic sunscreens should be used to prevent sun damage. To provide a controlled, daylight PDT environmental setting and remove the disadvantage of exposure to harmful ultraviolet radiation, SD-PDT has been investigated using an indoor daylight-simulating lamp. Wulf and co-workers reported that four different lamp candidates (18 W red-, 140 W red-, and 50 W white-light-emitting diode lamps and halogen lamps from 250 W slide projectors as well as 400 W overhead projectors for SD-PDT were able to photobleach a PPIX photosensitizer completely [39]. Calzavara-Pinto et.al. revealed that SD-PDT using a lamp with an output confined to the red waveband (630 ± 5 nm) and a polychromatic white LED lamp (400-700 nm) can represent a valid therapeutic method for Actinic cheilitis [41]. In our study, the SD-PDT effect can be obtained under the irradiation of a sunlight Xenon lamp with an emission spectral range of 380 nm to 700 nm. Hence, a sunlight Xenon lamp (380-700 nm) is also a useful lamp candidate for SD-PDT. However, in our study, we did not evaluate and compare the SD-PDT effect of Ag-modified TiO2 under other lamp sources. In further studies, more detailed comparative research may be needed to obtain a better SD-PDT anti-tumor therapeutic effect.

Discussion
Photodynamic therapy is an effective therapeutic strategy for skin disease in clinical therapy [38]. However, it is often accompanied by severe adverse effects during treatment. A large number of patients are unable to continue treatment due to these adverse effects. In 2008, daylight PDT was first introduced as a less painful, outdoors alternative to conventional PDT, with similar clinical effectiveness [39]. However, daylight PDT efficacy was often dependent on weather conditions. For example, in the U.K., daylight PDT is practical between the months of March or April and September or October, when the temperature is above 10 • C in the day (from 9:00 to 18:00) and the fluence rate reaches 130 W/m 2 [40]. In addition, to avoid patient exposure to harmful wavelengths of ultraviolet radiation during daylight PDT, organic sunscreens should be used to prevent sun damage. To provide a controlled, daylight PDT environmental setting and remove the disadvantage of exposure to harmful ultraviolet radiation, SD-PDT has been investigated using an indoor daylightsimulating lamp. Wulf and co-workers reported that four different lamp candidates (18 W red-, 140 W red-, and 50 W white-light-emitting diode lamps and halogen lamps from 250 W slide projectors as well as 400 W overhead projectors for SD-PDT were able to photobleach a PPIX photosensitizer completely [39]. Calzavara-Pinto et.al. revealed that SD-PDT using a lamp with an output confined to the red waveband (630 ± 5 nm) and a polychromatic white LED lamp (400-700 nm) can represent a valid therapeutic method for Actinic cheilitis [41]. In our study, the SD-PDT effect can be obtained under the irradiation of a sunlight Xenon lamp with an emission spectral range of 380 nm to 700 nm. Hence, a sunlight Xenon lamp (380-700 nm) is also a useful lamp candidate for SD-PDT. However, in our study, we did not evaluate and compare the SD-PDT effect of Ag-modified TiO 2 under other lamp sources. In further studies, more detailed comparative research may be needed to obtain a better SD-PDT anti-tumor therapeutic effect.
In this study, an investigation to improve the strategy to increase the simulatingdaylight response of existing photosensitizers is the main purpose of the research that we want. TiO 2 is a potent oxygen radical generator. However, it is limited in SD-PDT by the necessity to use ultraviolet irradiation with low tissue penetration and its harmful impact on the human body. To maximize the visible light absorption of TiO 2 , inorganic compounds were usually doped to the TiO 2 during their preparation, because this process can narrow the bandgap in the TiO 2 nanoparticle's structure and decrease the necessary activation energy. Among these inorganic compounds, noble metals (such as gold (Au), silver (Ag), platinum (Pt), and palladium (Pd)) were used to dope TiO 2 , one after another [33]. All absorption ranges of TiO 2 were shifted to longer wavelengths and enhanced photocatalytic activities under visible light were obtained to different degrees after doping. However, compared with the other noble metals used, Ag has been regarded as a better candidate due to its higher catalytic activity and ROS generation ability [41,42]. Hence, Ag-doped TiO 2 may be suitable for daylight PDT or SD-PDT. Unfortunately, there are few study reports that shows that Ag-doped TiO 2 is used in daylight PDT or SD-PDT. However, Alshamsan et.al. revealed that Ag-doped TiO 2 has the potential to selectively kill cancer cells while sparing normal cells through ROS generation in HepG2 (human liver cancer cell line) [43]. It gave us a reason to conduct research and evaluate the SD-PDT effect of Ag-doped TiO 2 on melanoma. In this study, the results showed that the limited photochemical activity and SD-PDT effect of TiO 2 could be improved significantly through doping Ag to the TiO 2 . In addition, our results showed that the degree of the improvement photochemical activity was independent of the concentration introduction of Ag into TiO 2 . This may be caused by the synthesized Ag-doped TiO 2 complex having different light responses with different concentrations of Ag under simulated-daylight irradiation. This change in light response was not entirely dependent on the doped Ag concentration. Lu and co-workers measured Ag-doped TiO 2 photocatalysts with different concentrations of Ag (1-5%) in their previous study. They reported that 2% Ag-doped TiO 2 had the highest photocatalytic activity under ultraviolet radiation and 5% Ag-doped TiO 2 had the highest photocatalytic activity under solar light [40]. Hence, the introduction of the different concentrations of Ag into TiO 2 may cause the different changes in light response. Generally, in our study, Ag-doped TiO 2 with a certain concentration of Ag efficiently improved TiO 2 photochemical activity compared with TiO 2 .
Besides the Ag-doped TiO 2 complex, TiO 2 -coated Ag nanoparticles have found applications in many fields because they can combine the surface plasmon resonance properties of a Ag core and the photoactivity of the TiO 2 shell [44]. Tunable optical properties can be obtained through a change in the ratio of the core radius and shell thickness. Hence, Ag-core TiO 2 was also usually used to increase the optical absorption of TiO2 and extend its absorption region to that of visible light. As with Ag-doped TiO 2 , there are few study reports that show that Ag-core TiO 2 is used in daylight PDT or SD-PDT, and the improvement in the photocatalysts' effect was not compared directly between Ag-doped TiO 2 and Ag-core TiO 2 in any other study. To find a better improvement strategy to increase the simulating-daylight response of a TiO 2 photosensitizer, the improvement in the photocatalytic activity and SD-PDT effect induced by Ag-doped TiO 2 and Ag-coreTiO 2 was compared. The results showed that the described TiO 2 modification method based on Ag significantly increased the photochemical properties of TiO 2 In addition, the synthesized Ag-doped TiO 2 was found to be a promising agent for treating melanoma using daylight PDT, and doping Ag to TiO 2 is the optimal enhanced strategy.
Several studies revealed that the introduction of Ag into TiO 2 improves TiO 2 's photochemical activity due to two mechanisms. (1) Ag can act as an electron acceptor to increase the separation efficiency of a photogenerated electron-hole pair because its Fermi level is below the conduction band of TiO 2 ; (2) The generation of a local surface plasmon resonance effect extends the visible light absorption range and increases the photocatalytic efficiency of TiO 2 [45]. Hence, in this study, first principles analysis was performed for Ag-doped TiO 2 and the discrete dipole approximation for the Ag-core TiO 2 was calculated. The results showed that a new shallow acceptor impurity level appeared in the energy band structure of Ag-doped TiO 2 , which decreased the recombination of photo-generated electrons and holes and the energy needed for the excitation of electrons. This expanded the light response range of TiO 2 and made it more responsive to sunlight. A strong field enhancement effect was obtained at the interface between the TiO 2 shell and the Ag core of Ag-core TiO 2 , which increased the amount of light captured by TiO 2 and improved its photochemical activity. These are consistent with the previously described mechanism. These further confirm the reliability of our research on this improvement strategy to increase the simulating-daylight response of a TiO 2 photosensitizer. Hence, Ag-doped TiO 2 is a promising photosensitizer agent for treating melanoma with daylight PDT.

Synthesis and Modification of TiO 2
Ag-doped TiO 2 : Nanosized TiO 2 was synthesized using the sol-gel process. Briefly, 5 mL of glacial acetic acid and 20 mL of absolute ethanol were added to 3 mL of ddH 2 O and stirred at room temperature for 30 min. A total of 5 mL of tetra-butyl titanate and 10 mL of absolute ethanol were mixed and added to the above solution. After stirring for 30 min, the solution was dispersed ultrasonically for 20 min, left to stand for 24 h at room temperature, and then dried at 80 • C for 12 h. The obtained dried product was fully milled and then cauterized to 450 • C in a muffle for 2 h. Purified TiO 2 was finally obtained. As with TiO 2 , 3 mL of 25 mg, 50 mg, 75 mg, or 100 mg AgNO 3 and 5 mL of glacial acetic acid were added in turn to 20 mL of absolute ethanol and stirred at room temperature for 30 min and then added to 15 mL of a tetra-butyl titanate solution containing 10 mL of absolute ethanol. After stirring, dispersing, drying, milling, and cauterizing, the Ag-doped TiO 2 with a mixing ratio of 1%, 2%, 3%, and 4% was obtained.
Ag-core TiO 2 : The Ag nanoparticles were produced utilizing the seed growth method. Initially, a 20 mL 1% sodium citrate solution diluted with 75 mL of ddH 2 O was stirred for 15 min at 70 • C. Next, 1.7 mL of 1% AgNO 3 and 2 mL of 0.1% NaBH 4 were added and stirred for 1 h at the same temperature. This process led to the creation of a 4 nm sized silver seed solution. Second, 2 mL of sodium citrate solution diluted with 80 mL of ddH 2 O was stirred for 15 min at 130 • C. Next, 10 mL of silver seed solution and 1.7 mL of AgNO 3 were added in turn, stirred for 1 h at the same temperature, and then centrifuged repeatedly. The purified Ag nanoparticles were yielded. Next, 0.3 mL of TiCl 3 , the different concentrations of 0.2 M sodium bicarbonate (0.9 mL, 1.1 mL, 1.3 mL, and 1.5 mL) and the silver nanoparticle solution were added in turn to 8 mL of ddH 2 O, stirred for 30 min, and then washed with ddH 2 O and absolute ethanol, respectively. Furthermore, 10 mL of N-butanol was added. The mixing solution was heated in oil baths at 100 • C for 10 min and then dried, milled, and cauterized. Finally, Ag-core TiO 2 with TiO 2 shells of different thicknesses were synthesized.

Characterization of Ag-Doped TiO 2 and Ag-Core TiO 2
The morphologies of Ag-doped TiO 2 and Ag-core TiO 2 were observed using transmission electron microscopy (TEM; JEM-2100, JEOL, Tokyo, Japan). Absorption spectra of Ag-doped TiO 2 and Ag-core TiO 2 were recorded using an ultraviolet-visible spectrophotometer (V-550 UV/VIS, JASCO, Tokyo, Japan). X-ray diffraction was conducted using an X-ray diffractometer (XPert Powder, PANalytical B.V. Netherlands). An energy dispersive spectrometer was used to observe the distribution pattern of various elements (Ag, Ti, and O) using TEM-EDS (JEM-2100 Plus, JEOL Ltd., Japan), operating with an accelerating voltage of 200 kV. The photocatalytic capability of Ag-doped TiO 2 (1%, 2%, 3%, and 4%) and Ag-core TiO 2 (5 nm, 10 nm, 15 nm, and 20 nm) were evaluated through the photocatalytic degradation of methylene blue under a simulated sunlight Xenon lamp with an emission spectral range of 380 nm to 700 nm. The irradiation time was 10 min at 665 nm. The absorption intensity was recorded using an ultraviolet-visible spectrophotometer (V-550 UV/VIS, JASCO, Tokyo, Japan).

Cell Viability Analysis
The cytotoxicity assay and phototoxicity assay to evaluate the safety and simulateddaylight PDT effect on melanoma cells (A375 cell line) were measured using a CCK-8 assay (Cell Counting Kit-8-allows for sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays). Briefly, A375 cells (8000/well) were seeded to sterile 96-well flat-bottomed plates and incubated overnight in a humidified incubator at 37 • C with 5% CO 2 . Diluted Ag-doped TiO 2 and Ag-core TiO 2 with the different concentrations (1 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 50 µg/mL) were added to corresponding cells in the 96-well flat-bottomed plates. After incubation for 6 h, the medium containing reagent was replaced by fresh cell culture medium. For the cytotoxicity experiment, the plates were then incubated for 24 h in a humidified incubator. In the phototoxicity experiment, the cells were irradiated with a sunlight Xenon lamp at 30 J/cm 2 for 15 min and then incubated for 12 h in a humidified incubator. To assess the influence of the irradiation dose on the phototoxicity effect, the cells were treated with 50 µg/mL of the Ag-doped TiO 2 or Ag-core TiO 2 previously mentioned. Then, they were irradiated with the daylight Xenon lamp at 10 J/cm 2 , 20 J/cm 2 , 30 J/cm 2 , 40 J/cm 2 , or 50 J/cm 2 for 15 min before being incubated for 12 h in a humidified incubator. Finally, all the treated cells were measured for absorbance levels at 450 nm using a microplate reader (Infinite M200 Pro., Tecan, Switzerland). The absorbance levels of cells were calculated as OD of treated group − OD of blank control group OD of control group − OD of blank control group × 100% OD is optical density.

Generation of ROS
ROS is an indirect factor to induce cell damage on PDT. Therefore, the ability of the generation of ROS was measured with a DCFH-DA probe using a Nikon eclipse Ti fluorescence microscope (Nikon, Japan). Briefly, 2.5 × 10 3 A375 cells were seeded to sterile 3.5 mL flat-bottomed plates and incubated overnight in a humidified incubator at 37 • C with 5% CO 2 . Then, the cells were treated with diluted Ag-doped TiO 2 , Ag-core TiO 2 , the synthesized TiO 2, and P25 for 6 h, washed with PBS twice, irradiated with the sunlight Xenon lamp at 40 J/cm 2 , incubated with 10 µmol/L DCFH-DA for 20 min at 37 • C in complete darkness, washed with PBS again, and then imaged using a FACScan system or detected using a fluorescence spectrophotometer under the excitation of 488 nm light. To quantify the ability of the generation of ROS, the fluorescence intensity of DCFH-DA was measured using a fluorescence spectrophotometer to detect the concentration of ROS in cells after being treated by Ag-doped TiO 2 , Ag-core TiO 2 , the synthesized TiO 2, and P25 under a simulated-sunlight Xenon lamp irradiation. The treated cells under the same conditions as above were harvested, incubated with 10 µmol/L DCFH-DA for 10 min at 37 • C in complete darkness, and then centrifuged, washed with PBS, and measured using a fluorescence spectrophotometer under an excitation of 488 nm light. In order to reveal the role of ROS more directly in simulated-daylight PDT induced by Ag-modified TiO 2 , inhibition tests were measured using a quenching agent of ROS (histidine). After being treated with the different agents containing TiO 2 , the cells were treated with 20 mM histidine for 30 min, washed with PBS, and then irradiated with the sunlight Xenon lamp. The cell activity was detected using CCK-8 analysis, as before.

First-Principles Analysis for Ag-Doped TiO 2
Based on crystallographic principles, the shape, electronic environment, and other parameters of the crystal cell will change when some atoms are substituted with allochthonous atoms in this cell. Therefore, the change in sunlight response induced by Ag-doped TiO 2 is most likely because some Ti atoms are substituted with Ag atoms. The energy band structure, the total density of states and the partial density of states, and the optical absorption properties were determined via density functional theory using the CASTEP code [47].

Discrete Dipole Approximation for Ag-Core TiO 2
It has been reported that the photocatalysis of TiO 2 could be enhanced using metal particle doping, polymer nanocomposites, core-shell nanoparticles, and so on, based on the localized surface plasmon resonance enhancement effect [48]. These electric field enhancement factors can be quantified and analyzed using simulations based on discrete dipole approximation [49]. Hence, theoretical mechanistic analysis of the change in sunlight response induced by silver-core TiO 2 in this study was employed using discrete dipole approximation simulations using the DDSCAT program.

Conclusions
To improve the limited effect of daylight PDT on melanoma due to the low daylight response of commonly used photosensitizers, Ag-mediated TiO 2 nanomaterials with different structures were synthesized, and then the improvement in the photocatalytic activity and PDT effect of these nanomaterials were compared. As per the findings, Ag effectively and significantly increased the photochemical activity and the PDT effect of TiO 2 under simulated-daylight irradiation. Ag-doped TiO 2 exhibited superior photocatalytic activity and a greater daylight-PDT-induced anti-tumor effect compared to Ag-core TiO 2 . To determine the mechanism, first principles analysis was conducted utilizing Ag-doped TiO 2 , whereas the discrete dipole approximation for Ag-core TiO 2 was calculated. The results showed that doping Ag into TiO 2 led to the formation of a new shallow acceptor impurity level in the energy band structure, which then enhanced the separation of electron-hole pairs, produced free conduction holes, reduced the recombination of photo-generated electrons and holes, and decreased the energy required for electrons to escape. This increased optical absorption in the range of 400-800 nm, which improved the photodamage effect of TiO 2 under simulated-daylight irradiation. The plasmonic near-field distribution increased due to the high refractive index of TiO 2 at the Ag-TiO 2 interface, which increased the amount of light captured by TiO 2 and enhanced the induction of the daylight PDT effect. In addition, when the thickness of the shell increased, the shell affected the movement of photo-generated electrons and holes, which decreased the overall photochemical activity. Overall, Ag proved to be highly effective in enhancing the photochemical activity and PDT effect of TiO 2 when exposed to simulated-daylight irradiation on melanoma. Thus, Ag-doped TiO 2 exhibits great potential as a photosensitizer agent for treating melanoma with daylight PDT.